Expression of pannexin 1 and 2 in cortical lesions from intractable epilepsy patients with focal cortical dysplasia

Abstract

Focal cortical dysplasia (FCD) is a major cause of intractable epilepsy in children however the mechanisms underlying the pathogenesis of FCD and FCD induced epilepsy remain unclear. Increasing evidence suggests that the large-pore ion channels, pannexin 1 (Panx1) and 2 (Panx2), are involved in epilepsy and brain development. In this study, we investigated the expression of Panx1 and Panx2 in surgical samples from patients with FCD type Ia (FCDIa), type IIa (FCDIIa), and type IIb (FCDIIb) and in age-matched autopsy control samples. We found Panx1 mRNA and protein levels were both increased in all these FCD samples. Immunohistochemical analyses revealed that Panx1 was mainly distributed in microcolumn neurons, dysmorphic neurons (DNs), balloon cells (BCs) and reactive astrocytes. Double-labeled staining showed that the Panx1-positive neurons were mostly glutamatergic DNs and occasionally GABAergic normal-appearing neurons. Importantly, the protein levels of Panx1 positively correlated with the frequency of seizures. Intriguingly, the Panx2 mRNA and protein levels were only upregulated in FCDIIb lesions and characteristically expressed on SOX2-positive multipotential BCs. Immunofluorescent experiments identified that Panx2-positive BCs mainly expressed the neuronal differentiation transcription factor MASH1 but not the immature glial marker vimentin. Taken together, our results established a potential role of the specific expression and cellular distribution patterns of Panx1 and Panx2 in FCD-associated epileptogenesis and pathogenesis.

Keywords: epileptogenesis; focal cortical dysplasia; pannexin 1; pannexin 2; pathogenesis.

Figure 1. Expression of Panx1 and Panx2 in FCD and CCx

A. Real-time PCR analysis of Panx1 mRNA expression in CCx (n = 8), FCDIa (n = 8), FCDIIa (n = 8) and FCDIIb (n = 8). * p < 0.05, CCx versus FCDIa, FCDIIa and FCDIIb. B. Panx2 mRNA expression in CCx and FCD groups. * p < 0.05, FCDIIb versus CCx, FCDIa and FCDIIa. Panx1 and Panx2 mRNA expression for each sample was normalized to β-actin. C. Representative immunoblot bands of Panx1 and Panx2 protein in total homogenates from CCx samples and FCD lesions. Molecular weight detected with Panx1 and Panx2 is shown at the expected bands of 47 kDa and 70 kDa. D. Densitometric analyses of Western blots. Values (Relative OD) are expressed as mean±SEM. * p < 0.05, ** p < 0.01. One way ANOVA.

Figure 2. Panx1 immunoreactivity (IR) in cortical lesions of FCD

A-B. Weak to moderate Panx1 IR in neurons (arrows in A), glial cells (double arrows in B) in gray matter (GM) and white matter (WM) of CCx. Insert in A refers NeuN positive neuron colabeled with Panx1. Insert in B refers GFAP positive astrocyte, not HLA-DR positive microglia colabeled with Panx1. C-D. Panx1 IR in FCDIa. Moderate to strong Panx1 IR in neurons (arrows in C), including in NeuN positive microcolumns (insert in C). Strong Panx1 IR in ectopic neurons (arrows in D) and glial cells (double arrows in D). E. Panx1 IR in FCDIIa. Moderate to strong Panx1 IR in DNs (arrows) and gliall cells (double arrows). Insert in E refers GFAP positive astrocyte colabeled with Panx1. F-H. Confocal images showing colocalization of Panx1 (green) with NF200 (red) in DN (arrow) in FCDIIa. I-J. Panx1 IR in FCDIIb. Moderate to strong Panx1 IR in DNs (arrows), BCs (arrowheads) and glial cells (double arrows). K. Merged images showing colocalization of Panx1 (green) with GFAP (red) in reactive astrocytes (double arrows) and BC (arrowhead, insert in K), but not in DN (arrow). L-N. Confocal images showing colocalization of Panx1 (green) with NF200 (red) in DN (arrow) and BC (arrowhead). O. Double labeling staining shows the HLA-DR (red) positive microglias (arrows) don't colocalize with Panx1 (green). 5-μm paraffin-embedded sections are counterstained with hematoxylin (A-E, I, J) or DAPI (F-H, K-O). Scale bars = (A-E, I, J) 30 μm, (F-H, K-O) 20 μm.

Figure 3. Double immunofluorescent staining of Panx1 in neurons in FCD

A-C. Representative confocal images show that strong Panx1 positive (green) DNs (arrows) colocalize with glutamate (Glu, red), and weak Panx1 positive (green) neurons (arrowheads) don't colocalize with Glu (red). D-F. Merged images show colocalization of Panx1 (green) with γ-aminobutyric acid (GABA, red) in the normal-appearing neurons (arrows), but not in the DN (arrowhead). G-I. Double labeling staining shows colocalization of Panx1 (green) with glutamic acid decarboxylase 67 (GAD67, red) in normal-appearing neuron (arrow), but not in the DNs (arrowheads) and BCs (double arrowheads). 5-μm paraffin-embedded sections are counterstained with DAPI. Scale bars = 30 μm.

Figure 4. Panx2 immunoreactivity (IR) in cortical lesions of FCD

A. Weak to moderate Panx2 IR in neurons (arrows) and glial cells (double arrows) in gray matter of CCx. B. Panx2 IR in FCDIa. Weak to moderate Panx2 IR in neurons (arrows), including in microcolumns and HNs (arrowhead, insert in B). C. Panx2 IR in FCDIIa. Weak to moderate Panx2 IR in DNs (arrows) and glial cells (double arrows). D-E. Panx2 IR in FCDIIb. Moderate to strong Panx2 IR in BCs (arrowheads in D). Weak (double arrows in D) and strong (arrow in D) Panx2 IR in DNs. Clusters of weak (triple arrowheads in E), moderate (double arrowheads in E) and strong (arrowhead in E) Panx2 IR positive BCs in FCDIIb. Weak to moderate Panx2 IR in glial cells (arrows in E). F-H. Double labeling staining shows colocalization of Panx2 (green) with NeuN (red) in DN (arrow) in FCDIIb. Some Panx2 positive BC colocalize with NeuN ( insert in E), and some are NeuN negative (arrowhead). I-J. Representative confocal images show the GFAP (red) positive BC (arrowhead) and reactive astrocytes (doublearrows) colocalize with Panx2 (green). GFAP negative DN (arrow) with stronger Panx2 IR than the GFAP positive cells. L. Merged images shows the HLA-DR (red) positive microglias (arrows) don't colocalize with Panx2 (green). 5-μm paraffin-embedded sections are counterstained with hematoxylin (A-E) or DAPI (F-L). Scale bars = (A-E) 30 μm, (F-L) 20 μm.

Figure 5. Double immunofluorescent staining of Panx2 in BCs in FCDIIb

A-C. Representative confocal images show some Panx2 positive (green) BCs (arrowheads) colocalize with SOX2 (red), and some are SOX2 negative (arrow, insert). D-F. Double labeling staining shows the Panx2 (green, arrowheads) don't colocalize with vimentin (vim, red, arrows) in BCs. G-I. Merged images show Panx2 (green) colocalize with MASH1 (red) in pyramidal neuron (double arrowheads) and BCs (arrowheads). 5-μm paraffin-embedded sections are counterstained with DAPI. Scale bars = 30 μm.

Figure 6. Correlation between the protein levels of Panx1 and different clinical variables in FCD

A, B. Scatter plot showing no significant correlation between the protein levels (relative optical density [OD]) of Panx1 and age at surgery (r = 0.135, P = 0.531), or duration of epilepsy (r = 0.182, P = 0.394). C. Scatter plot showing the significant positive correlation between the protein levels of Panx1 and seizure frequency (seizures per month), (r = 0.627. P = 0.001).